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Evaluation of the Effect of GM-CSF Blocking on the Phenotype And www.nature.com/scientificreports OPEN Evaluation of the efect of GM-CSF blocking on the phenotype and function of human monocytes Noushin Lotf1,2, Guang-Xian Zhang1, Nafseh Esmaeil2* & Abdolmohamad Rostami1* Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent cytokine that prompts the proliferation of bone marrow-derived macrophages and granulocytes. In addition to its efects as a growth factor, GM-CSF plays an important role in chronic infammatory autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. Reports have identifed monocytes as the primary target of GM-CSF; however, its efect on monocyte activation has been under-estimated. Here, using fow cytometry and ELISA we show that GM-CSF induces an infammatory profle in human monocytes, which includes an upregulated expression of HLA-DR and CD86 molecules and increased production of TNF-α and IL-1β. Conversely, blockage of endogenous GM-CSF with antibody treatment not only inhibited the infammatory profle of these cells, but also induced an immunomodulatory one, as shown by increased IL-10 production by monocytes. Further analysis with qPCR, fow cytometry and ELISA experiments revealed that GM-CSF blockage in monocytes stimulated production of the chemokine CXCL-11, which suppressed T cell proliferation. Blockade of CXCL-11 abrogated anti-GM-CSF treatment and induced infammatory monocytes. Our fndings show that anti-GM-CSF treatment induces modulatory monocytes that act in a CXCL-11-dependent manner, a mechanism that can be used in the development of novel approaches to treat chronic infammatory autoimmune diseases. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent cytokine that stimulates the proliferation of bone marrow-derived macrophages and granulocytes. Various cell types produce this cytokine, including activated T cells, monocytes/macrophages, B cells, NK cells, endothelial, epithelial, and fbroblasts cells1. GM-CSF has been identifed as a major cytokine in chronic infammatory autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA)2,3 GM-CSF plays a crucial role in RA progression and aug- ments infammatory immune responses in synovia4,5. Moreover, GM-CSF-producing CD4+ T cells in the blood and lesions of untreated MS patients correlate with disease severity6. We have shown that GM-CSF is necessary for the pathogenicity of T17 cells in experimental autoimmune encephalomyelitis, the prototypical animal model for MS7. GM-CSF exerts its function by binding to its receptor, which is composed of two diferent subunit α (CD116; GM-CSF Rα) and β chains (CD131; GM-CSF Rβ) with low and high afnity, respectively. Te alpha subunit is involved in ligand-specifc binding while the beta chain plays a central role in the signal transduction pathway8. GM-CSF signaling afects the survival and activation of myeloid cells, dendritic cell (DC) diferentiation and M1 macrophage phenotype polarization; it boosts antigen presentation, induces phagocytosis, recruits monocytes and other myeloid populations from bone marrow to circulation and promotes chemotaxis9,10. It has been recently demonstrated that CCR2+Ly6Chi infammatory monocytes are a target of GM-CSF in CNS autoimmunity by stimulating infammatory monocytes and their conversion into pathogenic macrophage-derived dendritic cells11–13. GM-CSF-activated monocytes migrate across the blood-brain barrier (BBB) and mediate BBB rupture by increasing expression of the endothelial adhesion molecules ICAM-1 and VCAM-114,15. GM-CSF also induces CCR2 expression in monocytes, which gives them an increased ability to cross the BBB. In EAE and MS, the CCR2-CCL2 axis has been previously shown to be a signifcant driver of infammatory leukocyte infltration into the CNS, and its activity positively correlates with disease pathogenesis16–18. Migration of leukocytes into the CNS is also mediated by CXCL9 and CXCL10 produced by glial cells19. Activated T lymphocytes in MS patients express CXCR3, which is the corresponding receptor of CXCL9, CXCL10, and CXCL-11 chemokines20. 1Department of Neurology, Thomas Jefferson University, Philadelphia, PA, USA. 2Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran. *email: [email protected]; [email protected] SCIENTIFIC REPORTS | (2020) 10:1567 | https://doi.org/10.1038/s41598-020-58131-2 1 www.nature.com/scientificreports/ www.nature.com/scientificreports It has been previously shown that while CXCL9 is a homing chemokine in the CNS, CXCL10, and CXCL-11 are induced afer infammation, and their role in infammation is less clear21–23. CXCL10 is involved in intrathecal infammation24. Interestingly, CXCL-11 is upregulated in MS patients afer IFN-β therapy and the decrease in the number of relapses may be linked to the increase in CXCR3 ligands in the serum of IFN-β-treated MS patients25. In this study, we analyzed the efect of GM-CSF on the phenotype and function of human monocytes. We found that GM-CSF treatment induces an infammatory phenotype in monocytes, while endogenous GM-CSF blocking is accompanied by an immunomodulatory phenotype. Further, GM-CSF blockade promoted CXCL-11 expression, and recombinant CXCL-11 inhibited the GM-CSF-induced proinfammatory impact of monocytes on T cells. Our fndings show that one of the mechanisms by which GM-CSF induces infammatory monocytes is the inhibition of CXCL-11 production and that this chemokine may be harnessed to suppress deleterious infam- matory responses observed in chronic infammatory diseases such as MS. Methods Isolation of human monocytes and culture treatments. All subjects gave informed consent before their participation in the current study. All human studies were approved by the Institutional Review Board (IRB) of Tomas Jeferson University, and all methods were performed in accordance with the relevant guidelines and regulations. Whole blood samples were collected from healthy donors and peripheral blood mononuclear cells (PBMCs) were enriched by gradient centrifugation in Ficoll. CD14+ monocytes were isolated by positive selection using magnetic beads following the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Te purity of cells was above 90%, measured by fow cytometry. Monocytes were seeded (1 × 106/ml) in 24-well plates and cultured in Iscove’s Modifed Dulbecco’s Medium (IMDM) (Gibco, Gaithersburg, MD, USA) supple- mented with 10% FBS, 1% penicillin/streptomycin antibiotic (Gibco), 2 mM glutamine and 2β- Mercaptoethanol (50 µg/ml, Gibco). Monocytes were activated with lipopolysaccharide (100 ng/mL, Sigma-Aldrich) for 18 h at 37 °C in the presence of recombinant human GM-CSF (10 ng/mL, R&D Systems, Minneapolis, MN, USA) or anti- GM-CSF (10 µg/mL, Biolegend, San Diego, CA). LPS-activated cells (mature monocytes) cultured with PBS were used as controls and culture of monocytes without LPS stimulation were considered as immature cells. RNA extraction, cDNA synthesis, and qPCR array. RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany), and the RNA concentration and quality were determined with Nanodrop (Thermofisher Scientific, Waltham, MA, USA). cDNA synthesis was performed from 1 µg of RNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Real-time PCR was performed using the TaqMan Array Human Immune Response (Applied Biosystems). Real-time PCR for CXCL-11 (Hs03003631_g1) was conducted™ according to the manufacturer’s instructions using TaqMan reagents (TermoFisher). Relative expression was calculated following the 2-ΔΔCT method, where 18 s (Hs03003631_g1) was considered the housekeeping gene. Flow cytometric analysis. For assessment of surface and intracellular cytokine expression, monocytes were collected afer 24 hours and stimulated for three hours with 50 ng/ml PMA (Sigma-Aldrich, St. Louis, MO,USA), 500 ng/ml ionomycin (Sigma-Aldrich), and 1 µg/ml GolgiPlug (BD Biosciences, San Jose, CA, USA). Cells were stained with anti-CD14 (M5E2, Biolegend), anti-CD16 (3G8, Biolegend.), anti-CD11b (ICRF44, Biolegend), anti-HLA-DR (L243, Biolegend), anti-CD80 (2D10, Biolegend), anti-CD86 (IT2.2, Biolegend), anti-CD83 (HB15e, Biolegend) and anti-PDL1(29E.2A3 Biolegend). Surface staining was performed for 20 min at 4 °C in the dark, and afer washing cells were fxed using 100 µl/tube fxation bufer at room temperature for 30 min (Termofsher Scientifc). Subsequently, the monocytes were permeabilized with 100 µl/tube Permeabilization Bufer (Termofsher Scientifc) and then stained with fuorochrome-conjugated antibodies for intracellular markers including anti-IL-10 (JES3-9D7, Biolegend), anti-TNF-α (MAb11, Biolegend) and anti-IL-1β (H1b-98, Biolegend), anti-IL-27 (B032F6, Biolegend) overnight at 4 °C in the dark. Also, the cells harvested from T- cells × monocytes co-culture treatments were stained for surface and intra- cellular markers with fuorochrome-conjugated antibodies including anti-CD3(SK7, Termofsher Scientifc), anti-CD4 (OKT4, Biolegend) anti-PDL-1 (29E.2A3, Biolegend), anti-IL-10 (JES3-19F1, Biolegend), anti-IFN-γ (B27, Biolegend), anti-RORɤt (Q21-559, BD Biosciense), anti-CD39 (A1, Biolegend). Samples were acquired on a BD FACS Aria Fusion (BD Biosciences) fow cytometry instrument and data ana- lyzed using Flowjo sofware 10. Te instrument calibration was examined before running the samples with BD FACSDiva™ CS&T research beads (CS&T research beads, BD Biosciences). Co-culture experiments.
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